Báo cáo khoa học: Thermodynamic analysis of Jun–Fos coiled coil peptide antagonists pdf

Thermodynamic analysis of Jun–Fos coiled coil peptide
antagonists
Inferences for optimization of enthalpic binding forces
Jonathan A. R. Worrall and Jody M. Mason
Department of Biological Sciences, University of Essex, Colchester, UK
Introduction
The transcriptional regulator activator protein-1 (AP-1)
generally consists of heterodimers of the Jun (e.g. cJun,
JunB, JunD) and Fos (e.g. cFos, FosB, Fra1, Fra2)
families of proteins. Different homologues combine to
form different heterodimers, which in turn have differ-
ent expression patterns depending on the tissue. AP-1
is responsible for the regulation of a number of key
genes that include cyclin D1 and interleukin-2, and is
Keywords
activator protein-1; coiled coil; isothermal
titration calorimetry; jun-fos; protein design
Correspondence
J. M. Mason, Department of Biological
Sciences, University of Essex, Wivenhoe
Park, Colchester CO4 3SQ, UK
Fax: +44 1206 872592
Tel: +44 1206 873010
E-mail:
(Received 23 August 2010, revised
12 November 2010, accepted 7 December
2010)
doi:10.1111/j.1742-4658.2010.07988.x
Dimerization of the Jun–Fos activator protein-1 (AP-1) transcriptional reg-
ulator is mediated by coiled coil regions that facilitate binding of the basic
regions to a specific promoter. AP-1 is responsible for the regulation of a

number of genes involved in cell proliferation. We have previously derived
peptide antagonists and demonstrated them to be capable of binding to the
Jun or Fos coiled coil region with high affinity (K
D
values in the low nM
range relative to lM for the wild-type interaction). Use of isothermal titra-
tion calorimetry combined with CD spectroscopy is reported to elucidate
the thermodynamic parameters that drive the interaction stability of pep-
tide antagonists with their cJun and cFos targets. We observe that the free
energy of binding for antagonist–target complexes is dominated by the
enthalpic term, is opposed by unfavourable entropic contributions consis-
tent with reduced conformational freedom and that these values in turn
correlate well (r = )0.97) with the measured helicity of each dimeric pair.
The more helical the antagonist–target complex, the more favourable the
change in enthalpy, which is in turn opposed more strongly by entropy.
Antagonistic peptides are predicted to represent excellent scaffolds for fur-
ther refinement. By contrast, the wild-type cJun–cFos complex is domi-
nated by a favourable entropic contribution, owing partially to a decrease
in buried hydrophobic groups from cFos core residues and an increase in
the conformational freedom.
Structured digital abstract
l
MINT-8077649, MINT-8077677, MINT-8077771, MINT-8077789, MINT-8077811, MINT-
8077831: c-Jun (uniprotkb:P05412)andc-Fos (uniprotkb:P01100) bind (MI:0407)byisothermal
titration calorimetry (
MI:0065)
l
MINT-8077856, MINT-8077872, MINT-8077889, MINT-8077906, MINT-8077923, MINT-
8077940: c-Jun (uniprotkb:P05412)andc-Fos (uniprotkb:P01100) bind (MI:0407)bycircular
dichroism (

MI:0016)
Abbreviations
AP-1, activator protein-1; CANDI, competitive and negative design initiative; ITC, isothermal titration calorimetry; PCA, protein-fragment
complementation assay; PPI, protein–protein interaction.
FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS 663
connected to a number of cell signalling cascades. It
has consequently been demonstrated that AP-1 upreg-
ulation is involved in a number of diseases, including
cancer [1–3] bone disease (e.g. osteoporosis) and
inflammatory diseases such as rheumatoid arthritis and
psoriasis [4–6]. Thus, peptides capable of specifically
sequestering key components of AP-1, and that there-
fore prevent its function, show great promise as the
starting point for drugs to combat a number of dis-
eases. The native AP-1 dimer (Fig. 1) consists of a
transactivation domain, a basic domain, rich in lysine
and arginine residues, that is responsible for mediating
DNA binding and a coiled coil (leucine zipper) region
that is known to mediate dimerization of the two
chains. Developing rules that can assist in the discov-
ery of new binding partners for coiled-coil-containing
proteins therefore has great potential for influencing
biology by elucidating stable and specific protein–pro-
tein interactions (PPIs) [8]. We have consequently
derived several peptides, based upon the coiled coil
regions of AP-1, that are able to bind to the corre-
sponding coiled coil regions of key AP-1 homologues
and prevent them from binding to DNA via their basic
region. Thus, these antagonists have the potential to
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Fig. 1. (A) The structure of the native DNA-
bound cJun–cFos AP-1 bZIP domain (PDB
coordinates 1FOS) [7] containing the bZIP
region of the two proteins. cJun is shown in
red and cFos in blue. The ‘basic’ N-terminal
regions are rich in arginine and lysine and
are responsible for scissor gripping the DNA
upon recognition of their cognate binding
sequence (TGACTCA). C-terminal of this
basic region is the leucine zipper (coiled coil)
region that is responsible for mediating
dimerization of the two chains, and is there-
fore the focus of this study. The figure cre-
ated using
PYMOL (DeLano Scientific; http://
pymol.sourceforge.net/). (B) A helical wheel
representation highlighting the interaction
patterns for the various heterodimers. Resi-
dues for cJun (left) and cFos (right) are col-
oured black. Residues for JunW, JunW
CANDI
and cJun(R) that differ from those of cJun
are shown as blue, green and red,
respectively. Similarly, residues for FosW,
FosW
Core
and FosW(E) that differ from
those of cFos are shown as blue, green and
red, respectively.

Coiled coils and ITC J. A. R. Worrall and J. M. Mason
664 FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS
sequester these proteins as nonfunctional heterodimers
to prevent binding to native partners. The first of these
peptides was generated by semirational design using
the native binding partner as a scaffold. Degenerate
codons important in dimerization were introduced and
a protein-fragment complementation assay (PCA)
[9,10] was undertaken to screen the resultant library
and single out peptide sequences capable of generating
an interaction with the target protein. This ensured
that only library members that bound to the target
generated colonies under selective conditions. Growth
competitions then ensured that only those PPIs of
highest affinity were enriched. The peptides, JunW and
FosW, bound to cFos and cJun, respectively, with
much higher interaction stability than the parent pro-
tein [11]. In order to increase the specificity of PCA-
generated PPIs, we incorporated a competitive and
negative design initiative (CANDI) into the screen.
CANDI is used to ensure that the energy gap between
desired and nondesired complexes is maximized and
works by including sequences competing for an inter-
action with either the target and ⁄ or the library member
in the bacterial selection [12,13]. Library members that
bind to the competitor, are promiscuous in their bind-
ing selection or cannot compete with the competitor–
target complex are subsequently removed from the
bacterial pool. Using the PCA–CANDI technique, we
generated a peptide, JunW

CANDI
, that is specific for
cFos even in the presence of a cJun competitor. This
is in sharp contrast to JunW, which binds with high
affinity to both cJun and cFos. This study offers the
possibility to look at the underlying thermodynamic
signature behind these two binding events. Libraries
based on the cJun–FosW peptide have also been cre-
ated with both core and electrostatic semirandomiza-
tions. Using competitive growth competitions, it was
found that the winner of the core randomization,
FosW
Core
, was able to bind to cJun specifically in the
presence of competing Fos homologues [14]. The
FosW
Core
library was based upon FosW and con-
tained 12 residue options (codon NHT = F, L, I, V,
S, P, T, A, Y, H, N or D) at four of five a position
residues. This study reflected the fact that core resi-
dues impose large energetic changes, with consequent
growth competitions, suggesting that they also have
the ability to impart specificity in instances where
electrostatic options are insufficient. Finally an elec-
trostatically enhanced dimer, cJun(R)–FosW(E), has
been previously studied to dissect the free energy of
binding into its component steps, and was found to
have achieved increased equilibrium stability as a
result of large decrease in the dissociation rate of the

complex [15].
Thermodynamics of binding
To enable us to address the question of a common
underlying mechanism by which all of these antago-
nists achieve high interaction affinity, we decided to
use CD data and isothermal titration calorimetry
(ITC) to split the free energy of binding into its com-
ponent parts, the enthalpy (DH) and the temperature
multiplied by the entropic contribution (TDS) accord-
ing to the relationship:
DG
bind
¼ DH À TDS ð1Þ
Where a negative DG
bind
value represents a sponta-
neous reaction that is favourable, DH represents the
strength of the target–antagonist complex relative to
those of the solvent and includes electrostatic bonds,
van der Waal’s interactions and hydrogen bond forma-
tion. A negative DH value is representative of a
favourable enthalpic contribution to the reaction. By
contrast, a positive TDS value represents a favourable
entropic contribution. Favourable entropy can come
from hydrophobic interactions that release water mole-
cules upon their formation as well as minimal loss in
conformational freedom. Although binding affinity can
be optimized by either enthalpic or entropic improve-
ments, so long as they are not compensated for by
opposite entropic or enthalpic changes [16,17], optimi-

zation of the binding energy via a negative enthalpic
term is favoured. However, optimizing noncovalent
bonds is extremely difficult to achieve by rational
design, because it is often accompanied by entropy
compensation. By studying a range of antagonists that
have been designed or selected by enriching the highest
affinity binding partners from libraries that target cJun
and cFos, it is anticipated that we can split the free
energy of binding into its thermodynamic components
to investigate whether there is a thermodynamic profile
that is common to all of these molecules.
Results
We used ITC to extract the thermodynamic parameters
that make up the overall free energy of binding (DG
bind
)
for our antagonist–peptide complexes. The antagonists
(see Table 1 for sequences and Fig. 2 for example ITC
profiles) have previously been shown to be capable of
sequestering cJun or cFos using a variety of techniques,
including CD thermal denaturation studies [11,12,20],
kinetic folding studies [15,21] and native gel analysis
[12,15]. We observe that the enthalpic component is
strongly favoured for our antagonist–target complexes
and that the change in entropy is unfavourable. How-
ever, in contrast to Seldeen et al. [18], we observe that
J. A. R. Worrall and J. M. Mason Coiled coils and ITC
FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS 665
the overall free energy of binding for the wild-type leu-
cine zipper complex is driven by a strong entropic com-

ponent. Moreover, as is the case for the parent AP-1
leucine zipper, our antagonists are predicted to form a
helical structure that gives rise to a coiled coil with
either the cJun or cFos target peptide. This structure is
maintained by core hydrophobic interactions, primarily
brought about by knobs into holes packing between
a–a¢ and d–d¢ residues, and from which the bulk of stabil-
ity arises. In addition, flanking electrostatic interactions
between g–e¢+1 core flanking residues are speculated to
play a primary role in specificity [22,23]. Together, both
of these types of interaction are predicted to give rise to a
favourable enthalpic transition upon binding. By con-
trast, the entropic term is largely dominated by the net
result of two opposing forces. The first, conformational
entropy (DS
conf
) results in a positive (unfavourable) net
contribution to the overall free energy of binding. DS
conf
arises from a reduction in conformational degrees of free-
dom of backbone and side chain atoms as the molecule
folds and gains structure. By contrast, desolvational
entropy (DS
solv
) contributes favourably to the net free
energy of binding and results from the release of water
molecules bound to regions of the target and antagonist
that become buried in fully formed complex.
Wild-type Jun–Fos leucine zipper region
The native coiled coil region of this human transcrip-

tional regulator produces a relatively weak interaction,
as has been well documented [11,12,21]. Addition of
DNA and other factors such as disulfide bridges
[24,25] and additional flanking regions [18,26–28] have
been shown to increase the stability of the complex. In
this analysis, however, we have focused entirely on the
unmodified coiled coil region of the wild-type AP-1
protein. This coiled coil dimerization motif is 4.5 hept-
ads in length. We find that the free energy of binding
is driven predominantly by a favourable entropy (TDS;
5.32 kcalÆmol
)1
), with only a very small enthalpic con-
tribution (DH; )0.82 kcalÆmol
)1
) to binding at 293 K.
The favourable entropy term arises mainly from desol-
vation effects which outweigh the unfavourable confor-
mational penalty. This is consistent with an observed
weak enthalpic contribution to the free energy of bind-
ing. Indeed, the free energy of binding is 2–3 kcalÆmol
)1
less than any of the antagonist–cJun or antagonist–
cFos complexes. ITC data collected from the leucine
zipper region of cJun and cFos correlate poorly with
the findings of Seldeen et al. [18] (see Tables 1 and 2).
We believe that their data overestimate the free energy
of binding for the leucine zipper region in the absence
of DNA. One possibility could be the use of a fusion
construct with a (His)

6
-tag and Trx-tag included to
necessitate purification and solubility of the cJun ⁄ cFos
leucine zippers. Seldeen et al. noted that these addi-
tional units were not anticipated to interact with the
bZIP domains of Jun and Fos.
Our ITC data on the stability of the cJun–cFos
interaction correlate well with thermal melting data
(see Table 2 and [11]), chemical denaturation data [12]
and earlier studies that have probed these regions [11]
(and references therein). In addition, both the bZIP
coiled coil prediction algorithm and the base-optimized
weights method of in silico coiled coil stability predic-
tion anticipate the measured stability of all of our
coiled coils pairs with reasonable accuracy, giving us
confidence in the reliability of our data. In addition,
Table 1. Peptide sequences and the sequences used by Seldeen et al. [18], which lack N and C capping motifs and contain an 11.7 kDa thi-
oredoxin motif fused to the N-terminus and a hexahistidine tag at the C-terminus, separtated by thrombin cleavage sites.
Name
Sequence
abcdefg abcdefg abcdefg abcdefg abcd
cJun AS IARLEEK VKTLKAQ NYELAST ANMLREQ VAQL GAP
cFos AS TDTLQAE TDQLEDE KYALQTE IANLLKE KEKL GAP
FosW AS LDELQAE IEQLEER NYALRKE IEDLQKQ LEKL GAP
JunW AS AAELEER VKTLKAE IYELQSE ANMLREQ IAQL GAP
JunW
CANDI
AS AAELEER AKTLKAE IYELRSK ANMLREH IAQL GAP
FosW
Core

AS IDELQAE VEQLEER NYALRKE VEDLQKQ AEKL GAP
cJun(R) AS IARLRER VKTLRAR NYELRSR ANMLRER VAQL GAP
FosW(E) AS LDELEAE IEQLEEE NYALEKE IEDLEKE LEKL GAP
LZ (cJun)
a
Trx-IARLEEK VKTLKAQ NSELAST ANMLREQ VAQLKQK-(His)
6
LZ (cFos)
a
Trx-TDTLQAE TDQLEDE KSALQTE IANLLKE KEKLEFI-(His)
6
a
Seldeen et al. [18,19] generated 28mers with peptides fused to an 11.7 kDa N-terminal thioredoxin (Trx) tag to assist with solubility and
expression, as well as a C-terminal (His)
6
-tag. Both tags were additionally separated by thrombin sites (LVPRGS) which upon cleavage
caused significant destabilization of the peptides. Their experimental conditions (50 m
M Tris, 200 mM NaCl, 1 mM EDTA and 5 mM b-mercap-
toethanol at pH 8) varied from this study.
Coiled coils and ITC J. A. R. Worrall and J. M. Mason
666 FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS
the comparatively low level of helicity (both measured
and predicted) for cJun, cFos and cJun–cFos (see
Table 3) supports the notion that this wild-type inter-
action is relatively modest in stability. Collectively,
previous studies on cJun–cFos leucine zipper pairs
have implied an interaction that is unstable
(T
m
=16°C [11], DG

bind
= 5.5 kcalÆmol
)1
[21]) at
physiological temperatures, which is considered impor-
tant in ensuring that the transcription factor is not
constitutively active in vivo. Rather, weak binding per-
mits the complex to extend its helicity into the basic
regions while either binding to or dissociating from the
DNA.
Table 2. Thermodynamic data from ITC, thermal melting analysis and bZIP K
D
values are shown as derived using ITC data, and in parentheses from data taken using the midpoint of the
transition from thermal denaturation profiles (and fit as temperature as a function of lnK
D
, with the fit lnK
D
= aT + C where a is the gradient, T is the temperature in Celsius and C is the
intercept) and calculated at 20 °C. Note that Seldeen’s data were collected at 25 °C, although data were also collected at 20 °C and show entropy–enthalpy compensation; a lowering of
the contribution made by DH (from )23 to approximately )18) is compensated for by a reduction in the contribution from TDS (from )13.5 to approximately )8), resulting in almost no
overall change in DG
bind
()9.7 to approximately )10). A = data obtained using van’t Hoff plots extracted from thermal melts and extrapolated to 20 °C. N ⁄ A, not available.
cJun–cFos [18] cJun–cFos cJun–FosW cJun–FosW
Core
cFos–JunW cFos–JunW
CANDI
cJun(R)–FosW(E)
NN⁄ A 1.06 (0.01) 0.65 (0.01) 0.95 (0.01) 0.69 (0.01) 0.80 (0.01) 0.78 (0.01)
K

B
(M
)1
) 1.6 · 10
7
3.76 · 10
4
(2.54 · 10
4
)
2.21 · 10
7
(4.28 · 10
6
)
7.9 · 10
5
(3.86 · 10
4
)
1.26 · 10
6
(9.15 · 10
4
)
8.31 · 10
5
(3.97 · 10
4
)

1.45 · 10
7
(2.37 · 10
6
)
DH (kcalÆmol
)1
) )23.21 (0.23) )0.82 (0.36) )10.55 (0.16) )11.94 (0.14) )13.9 (0.24) )14.8 (0.25) )27.0 (0.40)
TDS (kcalÆmol
)1
)[DH – DG] )13.52 (0.42) 5.32 (0.53) )0.68 (0.19) )4.03 (0.14) )5.72 (0.24) )6.86 (0.25) )17.4 (0.41)
DG
bind
ITC (kcalÆmol
)1
) )9.69 (0.26) )6.14 (0.39) )9.87 (0.11) )7.91 (0.03) )8.18 (0.04) )7.94 (0.03) )9.60 (0.10)
K
D
20 °C ITC (M
)1
thermal) 60 nM (25 °C) (N ⁄ A) 26.6 lM (324 lM) 45 nM (4 nM) 1.27 lM (20 lM) 0.79 lM (40 lM) 1.2 lM (0.45 lM) 69 nM (0.15 nM)
DG thermal (kcalÆmol
)1
)
A
N ⁄ A )5.5 )11.4 )8.6 )8.1 )8.5 )18.0
Measured T
m
N ⁄ A1663454444 98
a

bCIPA T
m
b
25 13 62 56 37 49 90
BOW
c
N ⁄ A 26.9 41.4 35.1 33.4 33.4 55.6
a
Extrapolated from fit using a restrained upper baseline based on alternative dimer thermal denaturation profiles.
b
Predicted thermal melting value based on sequence data using basic
coiled coil interaction algorithm (bCIPA).
c
Predicted interaction score according to base optimized weights (BOWs) [29].
Table 3. Helical calculations to assist in establishing whether the
peptide is representative of a coiled coil structure [30–32].
Peptides
(150 l
M Pt) h
222
⁄ h
208
Fraction
helical (ƒ
H
)
Averaged helicity
in % predicted by
Agadir (293 K)
cJun 0.53 14.6 3.7

cFos 0.65 17.3 3.5
FosW 1.02 43.7 26.2
JunW 1.01 41.7 17.0
JunW
CANDI
0.79 22.2 21.9
FosW
Core
0.74 26.6 10.2
cJun(R) 0.54 22.3 4.8
FosW(E) 0.45 17.2 7.9
cJun-cFos 0.75 25.0 3.6
cJun–FosW 1.00 40.0 15.0
cJun–FosW
Core
0.91 43.1 7.0
cFos–JunW 1.00 45.7 10.3
cFos–JunW
CANDI
0.97 48.4 12.7
cJun(R)–FosW(E) 1.00 88.0 6.4
The h222 ⁄ h208 ratios provide information on the likelihood of the
alpha-helix being in isolation or being found within a coiled coil
structure [30,31,33]. A ratio > 1.0 typically indicates the latter,
whereas a ratio of $ 0.9 or less indicates the presence of a helix in
isolation. For all dimeric pairs, except the wild-type structure (which
is known to interact with low affinity), the ratio is > 0.9, supporting
the formation of a coiled coil structure. Fraction helicity (ƒ
H
) can

be calculated as ƒ
H
=(h
222
) h
c
) ⁄ (h
222¥
) h
c
), where h
222¥
=
()44000 + 250T) · (1 – k ⁄ Nr) and h
c
= 2220 – (53 · T). In these
equations the wavelength-dependent constant k = 2.4 (at 222 nm),
Nr = the number of residues and T =20°C (293 K). Agadir [34–36]
severely underestimates helicity for many of the dimeric pairs,
most likely because it does not take into account the interhelical
interactions that assist with helix integrity in the dimeric pairs; it
considers only the helicity of individual helices in isolation. Thus,
the measured helicity is often higher than the values predicted
from the average of the two constituent helices by Agadir. Indeed,
in the most extreme case, cJun(R)–FosW(E), interhelical electro-
statics are particularly prominent. When not considered, these e ⁄ g
interactions would be grossly underestimated as merely the aver-
age of the two isolated constituent helices (6.4%). However, at
88% measured helicity, this ER pair associates to form the most
helical and indeed most stable coiled coil interaction in this study.

J. A. R. Worrall and J. M. Mason Coiled coils and ITC
FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS 667
Peptides designed to target cJun
FosW and FosW
Core
have both been designed to target
the cJun peptide. Both form dimeric complexes with
cJun that are much more stable than wild-type
(DG
bind
= )9.9 and )7.9 kcalÆmol
)1
relative to )6.1
kcalÆmol
)1
). For both antagonists, the majority of this
increased interaction stability is the result of a favour-
able enthalpy ()10.6 and )11.9 kcalÆmol
)1
relative to
)0.82 kcalÆmol
)1
; see Table 2), with the entropic
component opposing the binding process. Although
FosW has 2 kcalÆmol
)1
more interaction stability for
cJun relative to FosW
Core
, and its enthalpic contribu-

tion is 1.4 kcalÆmol
)1
less, the entropic penalty is
> 3 kcalÆmol
)1
less. Therefore the interaction is more
stable. The fact that the entropic term is much less
unfavourable than for cJun–FosW
Core
agrees well with
the predicted helical propensity of FosW and FosW
Core
;
both the measured helicity (taken using the 222 signal
and expressed as a fraction of maximal potential helici-
ty according to Hodges and co-workers [30,31] and
Shepherd et al. [33]), and calculated helicity according
to Agadir [34–36] predicts that FosW
Core
has approxi-
mately half the average helical content of FosW at
293 K [15,34–36]. However, upon binding to cJun,
both heterodimeric pairs display similar measured
helicity, suggesting that for cJun–FosW, DS
conf
and
DS
solv
almost cancel each other out. However, when
FosW

Core
binds cJun, the entropic contribution disfa-
vours the overall interaction stability. There is very lit-
tle increase in the predicted helicity of subunits upon
binding, suggesting that desolvation effects are out-
weighed by conformational entropy for this pair. By
contrast, for cJun–FosW, which has similar measured
helicity but very little unfavourable entropy, conforma-
tional entropy is likely to be comparable but with
increased desolvational entropy contributions. Thus,
residual water molecules, possibly resulting from an
additional alanine residue in the core region of the
cJun–FosW
Core
complex, may be responsible for gener-
ating a more unfavourable DS
solv
, although a strong
overall enthalpic term is maintained. This is consistent
with a library in which four of the five a¢ positions
were selected from twelve residue options [14] to give
an improved enthalpy of binding, over FosW.
Peptides designed to target cFos
JunW and JunW
CANDI
have both been selected using
PCA, but the latter has been generated to bind cFos
with increased specificity in the presence of a cJun
competitor, thus rendering the interaction stable and
specific [12]. Analysis of the ITC data informs that, in

agreement with thermal denaturation data, there is
almost no change in the free energy of binding. How-
ever, dissection of this value into its thermodynamic
components reveals JunW
CANDI
to have a slight
increase in enthalpy change upon binding cFos ()14.8
Fig. 2. Isothermal titration calorimetry (ITC) analysis of leucine zipper domain interactions between cJun and cFos, as well as their interac-
tion with peptide antagonist. (A) cFos into cJun, (B) cFos into JunW
CANDI
and (C) cJun into FosW
CORE
. The upper and lower panels show
raw data and data after baseline correction, respectively. During ITC experiments, $200–600 l
M of peptide A was injected in 30–40 · 5 lL
batches from the injection syringe into the cell, which contained 10–40 l
M peptide B. Both partners were in a 10 mM potassium phosphate
buffer, 100 m
M potassium fluoride at pH 7. Experiments were undertaken at 20 °C. The solid lines represent the fit of the data to the func-
tion based on the binding of a ligand to a macromolecule using Microcal (GE Healthcare, Uppsala, Sweden)
ORIGIN software [39].
Coiled coils and ITC J. A. R. Worrall and J. M. Mason
668 FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS
versus )13.9 kcalÆmol
)1
), suggesting that more non-
covalent bonds have been formed. However, the
enthalpy gain is offset by an equal opposing change in
the entropic term ()6.9 versus )5.7 kcalÆmol
)1

ÆK
)1
),
suggesting that the additional favourable enthalpic
interactions have not been matched by desolvation
effects, but have added a slight increase in helical pro-
pensity. This is in accordance with Agadir and mea-
sured helicity (see Table 3), which predicts JunW
CANDI
to have slightly higher helical propensity, contributing
to an unfavourable entropic contribution to the free
energy of binding.
cJun(R)–FosW(E)
This designed interaction was generated to investigate
the role of electrostatics in the folding of Jun–Fos-
based AP-1 coiled coils [15]. The dimer has a signifi-
cantly enhanced electrostatic (g ⁄ e) complement. This is
of particular interest in aiding future design rounds
because we have previously shown it to significantly
enhance dimeric stability as the result of a decrease in
the dissociation rate of the dimeric complex. In con-
trast to designing for increased rates of association,
this has considerable implications in the design of
effective inhibitors. Tailoring the dissociation rate
using kinetic design opens up the possibility to increase
antagonist efficacy by lengthening the time span that
the antagonist–target complex can endure [15,37,38].
The ITC data show that for this dimer there is a very
large enthalpic contribution to the interaction stability
()27 kcalÆmol

)1
) relative to the other PCA-selected
antagonists ()10.6 to )14.8 kcalÆmol
)1
) that is, in turn,
compensated for by an opposing but comparatively
small entropic penalty ()17.4 kcalÆmol
)1
ÆK
)1
). The rel-
atively modest helicity for Jun(R) and FosW(E) pep-
tides in isolation, measured by both helicity and
Agadir, would appear to suggest that conformational
entropy is not a major contributory factor in the effi-
cacy of this dimer. However, the measured helicity of
the heterodimer is very high (88%; see Table 3), and is
in stark contrast to the helical level predicted from
Agadir. This is because, in using Agadir, the helices
have been considered in isolation and averaged. How-
ever, in reality, the Arg–Glu salt bridges contribute
enormously to the integrity of the helical structure via
intermolecular electrostatic interactions, and in doing
so additionally contribute to a large and favourable
enthalpic term. This molecule, therefore, has a large
and unfavourable contribution from DS
conf
, in agree-
ment with the high level of measured helicity, and is
also likely to have a poor opposing entropic term from

DS
solv
because these additional core-flanking electro-
static e ⁄ g interactions are also likely to be heavily
solvated. Curiously, although the cJun(R)–FosW(E)
dimer is among the most stable of all those measured,
the ITC data do not predict the level of stability that
was observed from thermal melting data and kinetic
folding studies previously reported [14]. However, what
is clear is that the magnitudes of the opposing forces
are large relative to the other dimers studied and the
entropic barrier is surpassed by a strongly opposing
enthalpic contribution to give a very stable overall
interaction. It is conceivable that less direct methods
for determining the thermodynamic stability are not
always as reliable as direct thermodynamic methods of
measurement such as ITC. This may be particularly
true for instances where the enthalpic contribution to
binding is significant.
In addition to the predicted levels of helicity from
Agadir and the experimentally measured levels from
the CD data, we also monitored the ratio between the
two minima in ellipticity of the helical CD spectra (see
Table 3). Hodges and co-workers [30,31] previously
reported that a 222 ⁄ 208 of approximately < 0.9 typi-
cally represents an a-helix, whereas a ratio of > 1.0 is
indicative of a stable coiled coil interaction. We note
that according to this calculation only FosW and
JunW appear to form coiled coiled homodimers,
whereas all heterodimers generate ratios that are

> 0.9, except for cJun–cFos (0.75), which is known to
have a low binding affinity.
Discussion
We have used ITC as a tool to dissect the free energy
profile into its component parts for the binding of Jun–
Fos-based coiled coil dimers. ITC allows the complete
thermodynamic characterization of a bimolecular inter-
action without the need to label or tether. This study
included both the wild-type cJun–cFos coiled coil
dimer and a range of peptide antagonists that have
been designed to bind to and sequester either cJun or
cFos. Splitting the free energy of binding into its ther-
modynamic constituents is important in helping us to
elucidate the best way to design for antagonist efficacy.
For example, it has been reported that optimizing for
the most favourable enthalpic contribution to the free
energy of interaction might prove to be a valuable and
complementary addition to established tools for select-
ing and optimizing compounds in lead discovery,
owing to the fact that it is a direct method for monitor-
ing the number and ⁄ or strength of noncovalent bonds
being formed (or broken) between the target and
antagonist during complex formation [17]. It has, how-
ever, been argued that the enthalpic parameter is also
J. A. R. Worrall and J. M. Mason Coiled coils and ITC
FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS 669
more difficult to optimize than the entropic contribu-
tion to binding, because engineering bonds of the cor-
rect length and angle is notoriously difficult to achieve,
as is minimizing the degree of interaction between

polar groups and the solvent while ensuring that the
complex remains in solution. Likewise, it is difficult to
overcome enthalpy–entropy compensation, because an
engineered gain in enthalpy during bond optimization
is often compensated for by entropic loss as the confor-
mation becomes restricted. Thus, complexes in which
the binding energy is dominated by a favourable DH
term may be preferred in choosing which to select and
take forward for further refinement. Reassuringly, all
of our PCA-selected pairs have a strong enthalpic
contribution to the free energy of binding, with the
entropic component generally disfavoured. Thus, semi-
rational design combined with PCA enriches the most
efficacious binders by achieving an ethalpically driven
antagonist–target interaction. For coiled coils selected
from core and electrostatic libraries, a range of
intermolecular noncovalent interactions has been
selected to optimize the DH term, with the DS term
appearing to be less essential during the selection
process. We previously noted that a designed cJun(R)–
FosW(E) pair based on cJun–FosW formed a very
strong interaction and that the enhanced electrostatics
exerted their effect predominantly on the dissociation
rate [15]. We speculate that maximizing the enthalpic
contribution while reducing the dissociation rate of the
antagonist–target complex is an unexplored method for
increasing overall binding stability and antagonist
efficacy. Finally, we report on the strong correlation
(r = )0.97) that is observed between the experimen-
tally determined percentage helicity (calculated from

the ratio of the observed h
222
CD minima and the max-
imal calculated minima possible for a completely heli-
cal peptide of same length) and the change in entropy
and enthalpy taken from the ITC data (see Fig. 3).
Thus, as the measured helicity increases, so does the
magnitude of the entropic component that opposes
binding. In addition, we observe that as the unfavour-
able entropic term increases, the contribution made by
the enthalpic term also increases, meaning that an
equally striking relationship is found between observed
helicity and enthalpy, as would be predicted from
enthalpy–entropy compensation. The strength of these
two relationships suggests that one may be able to
monitor the CD spectra of known helical PPIs to assist
with the prediction of entopic and enthalpic contribu-
tions to the overall binding energy.
The importance of dissecting equilibrium stability to
investigate the kinetic contribution to the stability of
designed protein–ligand, and particularly protein–drug,
interactions is becoming an increasingly recognized area
of design [37,38,40]. Further work is required to study
the effect of this parameter on PPI specificity, but this
study highlights the need for thermodynamic analysis
to understand how key PPIs achieve interaction stabil-
ity and how this information might feed-forward to
assist with other parameters in future rounds of protein
design. This is likely to be useful in developing peptide
and peptidomimetic antagonists for lead discovery in

which early identification of hits is likely to vastly accel-
erate the path to lead discovery [41].
Experimental procedures
Protein preparation
Peptides were previously derived by either using semira-
tional design and selection with PCA or CANDI–PCA, or
were designed based on these previously selected structures.
Once the sequence of each peptide antagonist (see Table 1)
had been verified by DNA sequencing, they were purchased
as >90% pure from Protein Peptide Research Ltd
10
% Helicity vs TΔS
0
% Helicity vs ΔH
–10
–20
Energy change (kcal·mol
–1
)
–30
100
80
6040
20
0
Percentage helicity (Calculated from
θ
222
)
Fig. 3. Measured helical percentage plotted against both DH and

TDS associated with the binding event. Although there are only six
data points, both plots reveal a striking relationship (r = )0.97)
between these two parameters collected from different experi-
ments. The negative gradient indicates that as the helicity of the
dimeric pair increases, so too does the entropic penalty because
the chains adopt a more ordered conformation. This is more than
compensated for by increased enthalpic contributions, which also
provide an excellent correlation with measure helicity. The mea-
sured helical percentage values are taken from the CD data by
using the value in molar residue ellipticity for the mimima at
222 nm. The thermodynamic data are derived from ITC.
Coiled coils and ITC J. A. R. Worrall and J. M. Mason
670 FEBS Journal 278 (2011) 663–672 ª 2011 The Authors Journal compilation ª 2011 FEBS
(Fareham, UK) as Fmoc synthesized and amidated ⁄ acety-
lated and contained N- and C-capping motifs for improved
stability and solubility. Peptides were further purified where
necessary using reverse-phase HPLC. Peptide concentra-
tions were determined in water using absorbance at 280 nm
with an extinction coefficient of 1209 m
)1
Æcm
)1
[42] corre-
sponding to a Tyr residue inserted into a solvent exposed
b3 heptad position.
ITC measurements
ITC measurements were made using a Microcal VP-ITC
instrument and data were collected and processed using the
origin 7.0 software package. All measurements were carried
out at least twice. Briefly, all peptides were studied at 20 °C

in 10 mm potassium phosphate and 100 mm potassium fluo-
ride at pH 7. Peptide 1 (600 lL) was loaded into the syringe
at a concentration between 175 and 250 lm. Peptide 2
(1800 lL) was loaded into the cell at 10–40 lm. The peptide
in the syringe and cell were reversed to check that the results
were unaffected by this change. The experiment was
undertaken by injecting 5 lL · 40 injections of peptide 1
into the calorimetric cell. The change in thermal power as a
function of each injection was automatically recorded using
Microcal origin software [39] and the raw data were inte-
grated to yield ITC isotherms of heat release per injection as
a function of the Fos to Jun molar ratio. In general, the con-
centration of peptide 2 loaded into the cell was 30 · the
anticipated PPI K
D
and the concentration of peptide 1 in the
syringe was at least 20 · the concentration of peptide 2. No
precipitation of protein was observed in any of the experi-
ments undertaken. Following ITC measurements, the data
were fit to a one-site model:
qðiÞ¼ðnD HVPÞ=2Þ½1 þðL=nPÞþðK
d
=nPÞ
Àf½1 þðL=nP ÞþðK
d
=nPÞ
2
Àð4L=nPÞg
1=2
ð2Þ

where q(i) is the heat release (kcalÆmol
)1
) for the ith injec-
tion, n is the stoichiometry of heterodimerization, V is the
effective volume of protein sample loaded into the calori-
metric cell (1.46 mL), P is the total Jun concentration in
the calorimetric cell (lm) and L is the total Fos concentra-
tion in the calorimetric cell at the end of each injection
(lm). This model is derived from the binding of a ligand to
a macromolecule using the law of mass action (assuming a
1 : 1 stoichiometry) to extract the various thermodynamic
parameters [18], namely the apparent equilibrium constant
(K
d
) and the enthalpy change (DH) associated with hetero-
dimerization. The free energy change (DG
bind
) upon ligand
binding can be calculated from the relationship:
DG
bind
¼ÀRT ln K
D
ð3Þ
where R is the universal molar gas constant (1.9872 kcalÆ
mol
)1
ÆK
)1
), T is the absolute temperature in Kelvin

(293.15 K) and K
D
is in the dissociation constant of binding
with units of molÆL
)1
. Finally, the entropic contribution
(TDS) to the free energy of binding was calculated by rear-
ranging Eqn (1) using the derived values of DH and DG
bind
.
Acknowledgements
This work was supported by funding from the Well-
come Trust (Grant #DBB2800). In addition, the
authors wish to thank the Department of Biological
Sciences RCIF funding for the purchase of an isother-
mal titration calorimeter.
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